Methods and compositions for multiplex amplification of nucleic acids

ABSTRACT

A method is described for predetermining ratios of primer pairs present in a single reaction vessel so as to achieve approximately equimolar yield of products. The ratios are determined as a function of the length of the amplicon and the length of other amplicons being simultaneously tested. The primers may desirably be for p53 gene sequences.

RELATED APPLICATION

This application claims priority to U.S. Provisional Application, SerialNo. 60/050,405, filed on Jun. 20, 1997, the text of which is expresslyincorporated herein.

BACKGROUND OF THE INVENTION

The polymerase chain reaction (PCR) is a simple and versatile method toamplify in vitro a specific segment of DNA for subsequent study (Saikiet al., Science 230: 1350 (1985); Saiki et al., Science 235:487 (1985)).The PCR method has gained widespread use in biomedical research, and hasrevolutionized the accurate and early diagnosis of many inherited andacquired genetic disorders (Eisenstein, N. Engl. J. Med. 332:178(1990)), particularly those caused by point mutations or smallinsertions or deletions including sickle cell anemia (Saiki et al.,Science 230:1350 (1985)), hemophilia A (Kogan et al., N. EngL. J. Med.317:985 (1987)), Tay-Sach's disease (Myerowitz, Proc. Natl. Acad. Sci.USA 85:3955 (1988); Myerowitz et al., J. Biol. Chem. 263:18587 (1988)),cystic fibrosis (Riordan et al., Science 245:1066 (1989)), and manyothers. With PCR, it is also possible to detect heterozygotic carriersin recessive disorders.

Polymerase chain reaction (PCR) is used for a variety of purposes. PCRcan be used to amplify genomic DNA or other sources of nucleic acids foranalysis. It is often desirable to be able to achieve equimolar yieldsof different length amplicons when performing multiplex PCR or multiplePCR reactions. Having an approximately equimolar yield of amplicons isparticularly useful, for example, when approximately equalrepresentation of certain regions of genomic DNA amplified aftermultiplex PCR is desired. Prior to the methods of present invention,finding the appropriate experimental conditions useful to achieve thisresult has been difficult because PCR amplifies nucleic acids havingdifferent lengths with different efficiencies. The yield of longeramplicons is often less than the yield of shorter amplicons because ofthose differences in PCR amplification efficiency. FIG. 1 shows thedifference in yields that one might expect, for example, when startingwith equal primer concentrations used to amplify amplicons of varyinglengths: A, B, C. There is a continuing need in the art for methodswhich permit the amplification of different sequences with the sameefficiency so that approximately equimolar products result.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method ofperforming multiplex PCR which achieve approximately equimolar products.

It is another object of the invention to provide a set of primers foramplification of p53.

It is yet another object of the invention to provide a set of primersfor amplification of p53 to achieve approximately equimolar products.

It is still another object of the invention to provide a mixture ofprimers for performing multiplex PCR.

These and other objects of the invention are provided by one or more ofthe embodiments provided below. In one embodiment of the invention amethod of performing multiple polymerase chain reactions in a singlevessel is provided. The method comprises the steps of priming DNAsynthesis on a template in a vessel with at least two sets of primers.The primers are present in the vessel at a predetermined ratio which isdescribed by the formula:

C_(A)=C_(L)(L_(A)÷L_(L))²

C_(A) is the concentration of primers for an amplicon A. C_(L) is theconcentration of primer for the longest amplicon. L_(A) is the length ofthe amplicon A. L_(L) is the length of the longest amplicon.

Another embodiment provided by the invention is a method of performingmultiple polymerase chain reactions in a single vessel. The methodcomprises priming DNA synthesis on a genomic p53 template in a vesselwith ten sets of primers which amplify exons 2-11 of p53. The primersare shown in SEQ ID NO: ID NOS: 1-20. The primers are present in thevessel at the following ratio: exon 2 (89.4): exon 3 (26.9): exon 4(450): exon 5 (245.8): exon 6 (138.3): exon 7 (101.8): exon 8 (193.0):exon 9 (70.8): exon 10 (146.5): exon 11 (177.3).

According to still another embodiment of the invention a set of primersfor performing multiple polymerase chain reactions in a single vessel isprovided. The set comprises twenty primers having sequences as shown inSEQ ID NO: 1-20.

According to yet another embodiment of the invention a mixture ofprimers for performing multiplex polymerase chain reaction is provided.The primers are present in the mixture at a predetermined ratio to eachother. The ratio of the concentrations of the primers is described by:

C_(A)=C_(L)(L_(A)÷L_(L))²

wherein C_(A) is the concentration of primers for an amplicon A; whereinC_(L) is the concentration of primer for the longest amplicon; whereinL_(A) is the length of the amplicon A; and wherein L_(L) is the lengthof the longest amplicon.

The present invention thus provides the art with a method useful forperforming multiplex PCR. This method is particularly useful foramplification of multiple exons of p53. Moreover, a particular primerset useful for performing such multiplex PCR is also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the difference in yields that one might expect, forexample, when starting with equal primer concentrations used to amplifyamplicons of varying lengths: A, B, C.

FIG. 2 illustrates the relationship for given values X and L_(L), usingthe amplicons from different exons of the human p53 gene as an example.

DETAILED DESCRIPTION OF THE INVENTION

It is a discovery of the present invention that approximately equimolaryields of amplicons of varying lengths can be easily produced bymultiplex PCR. It has been determined that varying the primerconcentrations as a function of the lengths of amplicons yieldsapproximately equimolar amounts of amplicons of varying lengths. Therelationship between primer concentration and the length of amplicons isas follows:

C_(A)=C_(L)(L_(A)/L_(L))^(x)

wherein C_(A)=the concentration of primers for an amplicon A;

C_(L)=the concentration of primer for the longest amplicon;

L_(A)=the length of amplicon A;

L_(L)=the length of the longest amplicon; and

X is usually not zero and is often between one and three.

This relationship can be placed in a computer readable medium or be usedwith a computer system if desired.

FIG. 2 illustrates the relationship for given values X and L_(L), usingthe amplicons from different exons of the human p53 gene as an example.Using primer concentrations as set forth, for example in FIG. 2, oneskilled in the art can determine the optimum set of primerconcentrations to yield approximately equimolar yields of varying lengthamplicons in a multiplex or multiple PCR. Preferably, primers havingboth comparable base composition and comparable melting temperatures areused. Also preferably, Mg⁺² concentration, annealing temperatures, andcycling times of the PCR are optimized prior to choosing the desired setof primer concentrations in accordance with the present invention.

PCR techniques applicable to the present invention include inter aliathose discussed in PCR PRIMER: A LABORATORY MANUAL, Dieffenbach, C. W.and Dveksler, G. S., eds., Cold Spring Harbor Laboratory Press (1995).

The present application further provides primer sequences, primerconcentrations, and experimental conditions useful in the amplificationof the coding region of the human p53 gene. Particularly useful primersfor amplification of exons of the p53 gene are set forth in Table 1.

TABLE 1 p53 Primer Set 20 primers in 1 mM Tris-HC1, pH 7.4, 0.1 mM EDTA,sequences: Exon 2: 5′ -TCATGCTGGATCCCCACTTTTCCTCTTG-3′ (SEQ ID NO:1) 5′TGGCCTGCCCTTCCAATGGATCCACTCA-3′ (SEQ ID NO:2) Exon 3: 5′-AATTCATGGGACTGACTTTCTGCTCTTGTC-3′ (SEQ ID NO:3) 5′-TCCAGGTCCCAGCCCAACCCTTGTCC-3′ (SEQ ID NO:4) Exon 4: 5′-GTCCTCTGACTGCTCTTTTCACCCATCTAC-3′ (SEQ ID NO;5) 5′-GGGATACGGCCAGGCATTGAAGTCTC-3′ (SEQ ID NO:6) Exon 5: 5′-CTTGTGCCCTGACTTTCAACTCTGTCTC-3′ (SEQ ID NO:7) 5′-TGGGCAACCAGCCCTGTCGTCTCTCCA-3′ (SEQ ID NO:8) Exon 6: 5′-CCAGGCCTCTGATTCCTCACTGATTGCTC-3′ (SEQ ID NO:9) 5′-GCCACTGACAACCACCCTTAACCCCTC-3′ (SEQ ID NO:10) Exon 7: 5′-GCCTCATCTTGGGCCTGTGTTATCTCC-3′ (SEQ ID NO:11) 5′-GGCCAGTGTGCAGGGTGGCAAGTGGCTC-3′ (SEQ ID NO:12) Exon 8: 5′-GTAGGACCTGATTTCCTTACTGCCTCTTGC-3′ (SEQ ID NO:13) 5′-ATAACTGCACCCTTGGTCTCCTCCACCGC-3′ (SEQ ID NO:14) Exon 9: 5′-CACTTTTATCACCTTTCCTTGCCTCTTTCC-3′ (SEQ ID NO:15) 5′-AACTTTCCACTTGATAAGAGGTCCCAAGAC-3′ (SEQ ID NO:16) Exon 10: 5′-ACTTACTTCTCCCCCTCCTCTGTTGCTGC-3′ (SEQ ID NO:17)5′-ATGGAATCCTATGGCTTTCCAACCTAGGAAG-3′ (SEQ ID NO:18) Exon 11:5′-CATCTCTCCTCCCTGCTTCTGTCTCCTAC-3′ (SEQ ID NO:19)5′-CTGACGCACACCTATTGCAAGCAAGGGTTC-3′ (SEQ ID NO:20)

Table 2 shows particularly useful concentrations of the primers setforth in Table 1 for multplex PCR amplification using the experimentalconditions set forth in Table 3.

TABLE 2 Primer Concentrations in p53 Primer Set Values of X 2 Typicalvalues of C_(L) 450 nM Amplicon Length Primer Concs Longest 4 368 bp450.0 nM 5 272 bp 245.8 nM 8 241 bp 193.0 nM 11 231 bp 177.3 nM 10 210bp 146.5 nM 6 204 bp 138.3 nM 7 175 bp 101.8 nM 2 164 bp  89.4 nM 9 146bp  70.8 nM Shortest 3  90 bp  28.9 nM

TABLE 3 Multiplex PCR Start with 250 ng of Template DNA. PCR Componentsfor 100 ul PCR in 0.2 ml thin walled tubes: Stock Conc Final Conc for 1reaction Buffer (No Mg) 10 X 1 X 10.0 ul MgCl₂ 25 mM 2.5 mM 10.0 ul dATP10 mM 200 uM  2.0 ul dCTP 10 mM 200 uM  2.0 ul dGTP 10 mM 200 uM  2.0 uldTTP 10 mM 200 uM  2.0 ul Taq GOLD 5 U/ul 10 U  2.0 ul p53 Primer Set 20X 1 X  5.0 ul Water Human genomic 250 ng DNA Total Volume 100.0 ul FinalConcentrations in Buffer (No Mg) are 10 mM Tris-HCl (pH 8.3), 50 mM KClTaq GOLD is AmpliTaq Gold ™ from Perkin Elmer catalog #N808-0243 PCRCycles: 35 Cycles: 94 C. 10 min 94 C. 30 sec 60 C. 30 sec 72 C. 45 sec72 C. 10 min To visualize amplicons by gel Analysis: Visualize PCRproducts on 4% NuSieve Agarose Gel NuSieve ™ Agarose 3:1 is from FMCcatalog #50092 Load 5 ul of PCR + loading buffer Use 50 bp Ladder(Gibco/BRL catalog #10416-014) as size marker Run gel at 125 Volts for30 min. to 90 min. Expected PCR Products: Order in Gel: Amplicon LengthAmplicon Length Exon 2 164 bp Exon 4 368 bp Exon 3  90 bp Exon 5 272 bpExon 4 368 bp Exon 8 241 bp Exon 5 272 bp Exon 11 225 bp Exon 6 204 bpExon 10 210 bp Exon 7 175 bp Exon 6 204 bp Exon 8 241 bp Exon 7 175 bpExon 9 146 bp Exon 2 164 bp Exon 10 210 bp Exon 9 146 bp Exon 11 225 bpExon 3  90 bp

Using the methods and reagents provided herein, we achieved multiplexPCR amplification of coding regions shown of the human p53 gene inapproximately equimolar amounts. That desirable result was achieved in asingle-tube reaction. The achievement of such desirable results with theremarkable convenience of a single tube reaction further illustrates thecontribution to the art made by the present invention.

The methods and compositions of the present invention are useful invirtually any context in which equimolar yields of various PCR productsare desired. Such contexts include without limitation paternity testing,forensic analysis, genetic screening, polymorphism detection, andmutation analyses. The present invention can be used to amplify nucleicacids for all forms of sequence analysis known to those skilled in theart. Sequence analysis techniques includes, for example,dideoxy-sequencing and sequence analysis using high-density nucleic acidarrays: the GeneChip® probe arrays or VLSIPS™ technology of Affymetrix,Inc. High density nucleic acid arrays are discussed for example in Chee,M., Yang, R., Hubbell, E., Berno, A., Huang, X. C., Stem, D., Winkler,J., Lockhart, D. J., Morris, M. S., & Fodor, S. P., Science 5287,610-614 (1996), U.S. Pat. No. 5,445,934, and International PublicationNo. WO 95/11995 corresponding to PCT Application No. PCT/US94/12305.

The p53 gene and its protein product are discussed in Molecular Biologyof the Cell, 3rd Edition, Alberts, B., Bray, D., Lewis, J., Raff, M.,Roberts, K., and Watson, J. D., Garland Publishing (1994) at pages 889and 1284-1289.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of any appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

20 1 28 DNA Artificial Sequence Description of Artificial SequenceDerived from coding sequence of human p53 gene. 1 tcatgctgga tccccacttttcctcttg 28 2 28 DNA Artificial Sequence Description of ArtificialSequence Derived from coding sequence of human p53 gene. 2 tggcctgcccttccaatgga tccactca 28 3 30 DNA Artificial Sequence Description ofArtificial Sequence Derived from coding sequence of human p53 gene. 3aattcatggg actgactttc tgctcttgtc 30 4 26 DNA Artificial SequenceDescription of Artificial Sequence Derived from coding sequence of humanp53 gene. 4 tccaggtccc agcccaaccc ttgtcc 26 5 30 DNA Artificial SequenceDescription of Artificial Sequence Derived from coding sequence of humanp53 gene. 5 gtcctctgac tgctcttttc acccatctac 30 6 26 DNA ArtificialSequence Description of Artificial Sequence Derived from coding sequenceof human p53 gene. 6 gggatacggc caggcattga agtctc 26 7 28 DNA ArtificialSequence Description of Artificial Sequence Derived from coding sequenceof human p53 gene. 7 cttgtgccct gactttcaac tctgtctc 28 8 27 DNAArtificial Sequence Description of Artificial Sequence Derived fromcoding sequence of human p53 gene. 8 tgggcaacca gccctgtcgt ctctcca 27 929 DNA Artificial Sequence Description of Artificial Sequence Derivedfrom coding sequence of human p53 gene. 9 ccaggcctct gattcctcactgattgctc 29 10 27 DNA Artificial Sequence Description of ArtificialSequence Derived from coding sequence of human p53 gene. 10 gccactgacaaccaccctta acccctc 27 11 27 DNA Artificial Sequence Description ofArtificial Sequence Derived from coding sequence of human p53 gene. 11gcctcatctt gggcctgtgt tatctcc 27 12 28 DNA Artificial SequenceDescription of Artificial Sequence Derived from coding sequence of humanp53 gene. 12 ggccagtgtg cagggtggca agtggctc 28 13 30 DNA ArtificialSequence Description of Artificial Sequence Derived from coding sequenceof human p53 gene. 13 gtaggacctg atttccttac tgcctcttgc 30 14 29 DNAArtificial Sequence Description of Artificial Sequence Derived fromcoding sequence of human p53 gene. 14 ataactgcac ccttggtctc ctccaccgc 2915 30 DNA Artificial Sequence Description of Artificial Sequence Derivedfrom coding sequence of human p53 gene. 15 cacttttatc acctttccttgcctctttcc 30 16 30 DNA Artificial Sequence Description of ArtificialSequence Derived from coding sequence of human p53 gene. 16 aactttccacttgataagag gtcccaagac 30 17 29 DNA Artificial Sequence Description ofArtificial Sequence Derived from coding sequence of human p53 gene. 17acttacttct ccccctcctc tgttgctgc 29 18 31 DNA Artificial SequenceDescription of Artificial Sequence Derived from coding sequence of humanp53 gene. 18 atggaatcct atggctttcc aacctaggaa g 31 19 29 DNA ArtificialSequence Description of Artificial Sequence Derived from coding sequenceof human p53 gene. 19 catctctcct ccctgcttct gtctcctac 29 20 30 DNAArtificial Sequence Description of Artificial Sequence Derived fromcoding sequence of human p53 gene. 20 ctgacgcaca cctattgcaa gcaagggttc30

What is claimed is:
 1. A method for combining primers for anamplification reaction of a set of multiple amplicons including anamplicon A and an amplicon L, wherein amplicon L is the longest ampliconin said set, comprising: calculating primer concentrations using theformula: C_(A)=C_(L)(L_(A)÷L_(L))² wherein C_(A) is the concentration ofprimers for an amplicon A; wherein C_(L) is the concentration of primersfor the longest amplicon; wherein L_(A) is the length of the amplicon A;and wherein L_(L) is the length of the longest amplicon; combining theprimers in a reaction vessel at the concentrations determined using saidformula.
 2. The method of claim 1 wherein the multiple ampliconscomprise genomic DNA encoding p53.
 3. The method of claim 1 wherein themultiple amplicons comprise a cDNA encoding p53.
 4. The method of claim1 wherein the multiple amplicons comprise at least 2 exons of p53selected from the group consisting of exons 2-11.
 5. The method of claim1 wherein the multiple amplicons comprise at least 4 exons of p53selected from the group consisting of exons 2-11.
 6. The method of claim1 wherein the multiple amplicons comprise exons 2-11 of p53.
 7. Themethod of claim 4 wherein the primers are selected from those shown inSEQ ID NOS: 1-20.
 8. The method of claim 5 wherein the primers areselected from those shown in SEQ ID NOS: 1-20.
 9. The method of claim 6wherein the primers are shown in SEQ ID NOS: 1-20.
 10. The method ofclaim 9 wherein the primers are combined in the following ratios: exon 2(89.4): exon 3 (26.9): exon 4 (450): exon 5 (245.8): exon 6 (138.3):exon 7 (101.8): exon 8 (193.0): exon 9 (70.8): exon 10 (146.5): exon 11(177.3).
 11. A kit comprising a set of primers for performing multiplepolymerase chain reactions in a single vessel, comprising: twentyprimers having sequences as shown in SEQ ID NO: ID NOS: 1-20.
 12. Thekit of claim 11 wherein the molar ratio of the concentrations of theprimers is described by: C_(A)=C_(L)(L_(A)÷L_(L))² wherein C_(A) is theconcentration of primers for an amplicon A; wherein C_(L) is theconcentration of primer for the longest amplicon; wherein L_(A) is thelength of the amplicon A; and wherein L_(L) is the length of the longestamplicon.
 13. The kit of claim 11 wherein the molar ratio of the primersis: exon 2 (89.4): exon 3 (26.9): exon 4 (450): exon 5 (245.8): exon 6(138.3): exon 7 (101.8): exon 8 (193.0): exon 9 (70.8): exon 10 (146.5):exon 11 (177.3).